Dermopharmaceutically and cosmetically active oligopeptides

ABSTRACT

Oligopeptides and derivatives thereof, peptide analogs and derivatives thereof as well as pharmaceutically acceptable salts of these compounds, which correspond to general formula (I) 
                         
wherein
     R 1  represents H, —C(O)—R 7 , —SO 2 —R 7 , —C(O)—OR 7  or —C(O)—N(R 7 ) 2      R 2  represents, independent of one another, H or —(C 1 -C 4 )-alkyl,   R 3  and R 6  represent, independent of one another, —(CH 2 ) q —N(R 1 )R 8      R 4  and R 5  represent, independent of one another, —CH 2 —OR 2 , —CH(CH 3 )OR 8  or —CH 2 —CH 2 —OR 8      R 7  represents hydrogen, optionally substituted (C 1 -C 19 )-alkyl; optionally substituted (C 1 -C 19 )-alkenyl; phenyl-(C 1 -C 4 )-alkyl whose phenyl radical is optionally substituted with amino in the para position   R 8  represents H, —(C 1 -C 4 )-alkyl, —C(O)—R 7 , —C(O)—OR 7 , —C(O)—N(R 7 ) 2  or —SO 2 —R 7      X represents oxygen (—O—) or —NH—; or   XR 7 , with X=O, also represents the esters of α-tocopherol, tocotrienol or retinol or the carboxylic acid (with R 7 =H)   m, n, p represent, independent of one another, zero or 1 and
 
q in R 3  and R 6  represent, independent of one another, an integer from 1 to 4, with the provision that the following conditions do not simultaneously occur: R 4 =—CH(CH 3 )—OH and R 5 =—CH(CH 3 )—OH and R 6 =—(CH 2 ) 4 —NH 2 ; dermopharmaceutically and/or cosmetically active compositions containing at least one compound of formula (I).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/CH02/00587, filed May 8, 2003, which claims the benefit ofApplication No. CH 1986/01, filed Oct. 30, 2001, the contents of whichis incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to pharmaceutically, dermopharmaceuticallyand cosmetically active oligopeptides, to derivatives thereof, topeptide analogs and derivatives thereof as well as to the use thereof indermopharmaceutically and cosmetically active compositions.

BACKGROUND OF THE INVENTION

The basic mechanism of skin aging is known to take place in theextracellular matrix, the so-called basal lamella, when situated at theintersection between epithelium and connective tissue. Regeneration ofthe extracellular matrix is highly important as it considerablyinfluences the behaviour of those cells that are in contact with it, inparticular their growth, migration, proliferation, form and functions.An age-related reduction of collagen synthesis by fibroblasts occurs inthis extracellular matrix, the consequence of which being a reducednumber of chemical substances secreted by these cells. As skin proteinsconsist by approx. 80% of collagen, already a small natural decrease ofthe collagen concentration in the tissue may have clear consequences onthe mechanical and physiological properties of skin.

Katayama et al. (The Journal of Biological Chemistry, Vol. 268, No. 14,pages 9941-9944, 1993) found that the minimal subfragment sequence forstimulating collagen and fibronectin is represented by the pentapeptideLys-Thr-Thr-Lys-Ser. Sequences with four amino acids or less have aslighter or no stimulating effect.

The European patent application WO 00/15188 describes the effect of thepalmitoylated pentapeptide Palm-Lys-Thr-Thr-Lys-Ser as a component fortreating skin aging, accelerating wound healing and improving skinmoisturizing.

SUMMARY OF THE INVENTION

It has been found that selected, new oligopeptides and derivativesthereof as well as peptide analogs and derivatives thereof (in thefollowing referred to as “compounds of the present invention”) aresurprisingly highly pharmaceutically and/or cosmetically effective andare particularly appropriate for use in dermopharmaceutically and/orcosmetically effective compositions. The compounds of the presentinvention diffuse rapidly and in sufficient concentration through thecell membrane up to the intracellular site of action, where they lead toa clearly increased production of collagen and fibronectin. Thus, thecompounds of the present invention exert a surprisingly positive andstimulating effect on the extracellular matrix which decisivelyinfluences the mechanical and physiological appearance of skin. Inparticular, the compounds of the present invention induce a much morerapid and stronger stimulation of collagen synthesis than known so farfor other compounds from the state of the art. The quicker and strongerstimulation of collagen synthesis by the compounds of the presentinvention is probably the result of a synergic effect which is obtainedby reduction of the molecular mass, introduction of N-methyl groups inamino acids and modification of amino acids with, e.g., fatty acidesters. However, the present invention is not bound to this explanation.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is defined in the claims and relates in particularto selected, new oligopeptides and derivatives thereof, peptide analogsand derivatives thereof, as well as pharmaceutically acceptable salts ofthese compounds, thereby characterized that these compounds correspondto general formula (I)

whereinR¹ represents H, —C(O)—R⁷, —SO₂—R⁷, —C(O)—OR⁷ or —C(O)—N(R⁷)₂,R² represents, independent of one another, H or —(C₁-C₄)-alkyl,R³, R⁶ represent, independent of one another, —(CH₂)_(q)—N(R¹)R⁸,R⁴, R⁵ represent, independent of one another, —CH₂—OR², —CH(CH₃)OR⁸ or—CH₂—CH₂—OR⁸,R⁷ represents hydrogen, (C₁-C₁₉)-alkyl optionally substituted once orseveral times, independent of one another, with halogen, hydroxy,carboxyl, amino, mercapto, 1,2-dithiolanyl, and/or sulfo; optionallysubstituted (C₁-C₁₉)-alkenyl; phenyl-(C₁-C₄)-alkyl, whose phenyl radicalis optionally substituted with amino in the para position; orR⁸ represents H, —(C₁-C₄)-alkyl, —C(O)—R⁷, —C(O)—OR⁷, —C(O)—N(R⁷)₂ or—SO₂—R⁷X represents oxygen (—O—) or —NH—; orXR⁷, with X=O, also represents the esters of α-tocopherol, tocotrienolor retinol or the carboxylic acid (with R⁷=H),m, n, p represent, independent of one another, zero or 1 and q in R³ andR⁶ represents, independent of one another, an integer from 1 to 4, withthe provision that the following conditions do not simultaneously occur:R⁴=—CH(CH₃)—OH and R⁵=—CH(CH₃)—OH and R⁶=—(CH₂)₄—NH₂.

Preferably, the following conditions do not simultaneously occur:R⁴=—CH₂—OH or —CH(CH₃)—OH and R⁵=—CH(CH₃)—OH and R⁶=—(CH₂)₄—NH₂.Preferably, the following conditions do not simultaneously occur: R²=Hand R⁴=—CH₂—OH or —CH(CH₃)—OH and R⁵=—CH(CH₃)—OH and R⁶=—(CH₂)₄—NH₂.

The compounds of formula (I) comprise dipeptides, tripeptides,tetrapeptides and pentapeptides of the corresponding amino acids andderivatives thereof according to the definitions of R¹, R², R⁷ and R⁸,as well as corresponding peptide analogs.

The amino acid residues in general formula (I), which contain thesubstituents R³ or R⁶, are derived from lysine (Lys), ornithine (Orn),2,4-diaminobutyric acid (Dab) or 2,3-diaminopropionic acid (Dap) and areresidues of these amino acids or amino acid derivatives.

The amino acid residues in general formula (I), which contain thesubstituents R⁴ or R⁵, are derived from serine (Ser), threonine (Thr),homoserine [H₂N—CH((CH₂)₂—OH)COOH, (HSe)] and are residues of theseamino acids or amino acid derivatives.

The following table shows from which amino acids the compounds of theabove formula (I) are derived:

R¹(N(R²)CH(R³)C(O) N(R²)CH(R⁴)C(O) N(R²)CH(R⁵)C(O) N(R²)CH(R⁶)C(O)N(R²)CH(R⁴)C(X)R⁷ derived from: derived from: derived from: derivedfrom: derived from: Lysine (Lys) Serine (Ser) Serine (Ser) Lysine (Lys)Serine (Ser) Ornithine (Orn) Homoserine (HSe) Homoserine Ornithine (Orn)Homoserine (HSe) 2,4-Diaminobutyric Threonine (Thr) (HSe)2,4-Diaminobutyric Threonine (Thr) acid (Dab) Threonine acid (Dab)2,3-Diaminopropionic (Thr) 2,3-Diaminopropionic acid (Dap) acid (Dap)

The residue [R¹(N(R²)CH(R³)C(O)—] is preferably derived from lysine.

The residue [—N(R²)CH(R⁴)C(O)—] at position[—N(R²)CH(R⁴)C(O)—N(R²)CH(R⁵)C(O)—] is preferably derived fromthreonine.

The residue [—N(R²)CH(R⁵)C(O)—] is preferably derived from threonine.

The residue [—N(R²)CH(R⁶)C(O)—] is preferably derived from ornithine,2,4-diaminobutyric acid (Dab), and 2,3-diaminopropionic acid (Dap).

The terminal residue [—(N(R²)CH(R⁴)C(O)—XR⁷] is preferably derived fromserine.

The compounds of formula (I) preferably contain the following sequences:

Sequence 1: -Ser-Ser-Orn- Sequence 2: -Thr-Thr-Orn- Sequence 3:-Thr-Thr-Dab- Sequence 4: -Thr-Thr-Dap-

The compounds with the sequences -Ser-Ser-Orn-, Lys-Thr-Thr-Orn-Ser,Lys-Thr-Thr-Dab-Ser and Lys-Thr-Thr-Dab-Ser, as well as the sequencescorrespondingly derivatized with the substituents R¹, R², R⁷ and R⁸ areparticularly preferred.

Further preferred polypeptides enclosed by formula (I) are mentioned inthe text.

Preferred compounds of formula (I) are also those wherein m=zero, when nor p=zero.

The terms “peptides” and “oligopeptides” above all comprise naturallyoccurring amino acids, peptides and oligopeptides, respectively. Peptideanalogs mean synthetically modified amino acids, peptides andoligopeptides, respectively, e.g. with a methyl group at the nitrogenatom (CH₃—N═). Derivatives within the present invention particularlymean amino acids, peptides and oligopeptides, respectively, the terminalamino group or carboxyl group of which has been further converted, e.g.wherein the terminal carboxyl group has been esterified.

The compounds of formula (I) are particularly appropriate aspharmaceutical, dermopharmaceutical and/or cosmetic active ingredientsfor the preparation of pharmaceutically, dermopharmaceutically and/orcosmetically active compositions, in particular for increasing thecollagen and fibronectin production in human skin.

Moreover, the present invention relates to a method for preparing thecompounds of the present invention and salts thereof and their use aspharmaceutical, dermopharmaceutical and/or cosmetic active ingredients,as well as pharmaceutically, dermopharmaceutically and/or cosmeticallyactive compositions which contain at least one compound of the presentinvention.

Furthermore, the present invention relates to the use of the compoundsof the present invention for preparing a wound healing- andmoisturizing-stimulating drug as well as to a method for delaying ortreating skin aging, in particular the formation of wrinkles, whichcomprises applying a compound of the present invention and/or acomposition of the present invention on the skin.

The above used, general terms are defined as follows:

Halogen means chlorine, bromine or iodine, preferably fluorine.

Alkyl, as a group per se or as a structural element of an alkoxyfunction, comprises linear as well as branched alkyl groups. Examplesthereof are methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl,n-octyl, n-nonyl, n-undecanyl, n-dodecanyl, n-tridecanyl, n-hexadecanyl,n-heptadecanyl, n-octadecanyl or n-nonadecanyl as unbranched residuesand isopropyl, tert.butyl, isobutyl, sec.butyl, isoamyl as branchedresidues. R² and/or R⁸ as alkyl preferably means methyl, ethyl andpropyl, preferably methyl. If R⁷ as a part of R¹ means or contains analkyl residue, it preferably represents alkyl with 8 to 22 C atoms,preferably with 14 to 17 C atoms. If XR⁷ contains an alkyl residue, itpreferably means alkyl with 1 to 22 C atoms, preferably with 1 to 4 Catoms.

Alkenyl has the denotation of a mono- or poly-unsaturated, optionallysubstituted alkyl group, such as e.g. 8(Z)-heptadecenyl,8(Z),11(Z)-heptadecadienyl, 4(Z),7(Z),10(Z), 13(Z)-nonadecatetraenyl,8(Z)-11-hydroxyoctadecenyl.

α-Tocopheryl means (D)-, (L)- or(DL)-2,5,7,8-tetramethyl-2-(4′,8′,12′-trimethyltridecyl)-6-chromanyl.Tocotrienyl means any isomer of2,5,7,8-tetramethyl-2-(4′,8′,12′-trimethyl-3′,7′,11′-tridecatrienyl)-6-chromanyl.Retinyl means3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraen-1-yl.

The compounds of formula (I) together with acids can form mono- orpolyvalent, homogeneous or mixed salts, e.g. with inorganic acids, suchas hydrochloric acid, bromhydric acid, sulfuric acid or phosphoric acid;or with appropriate organic aliphatic saturated or unsaturatedcarboxylic acids, e.g. aliphatic mono- or dicarboxylic acids, such asformic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid,propionic acid, glycolic acid, succinic acid, fumaric acid, malonicacid, maleic acid, oxalic acid, phthalic acid, citric acid, lactic acidor tartaric acid; or with aromatic carboxylic acids, such as benzoicacid or salicylic acid; or with aromatic-aliphatic carboxylic acids,such as mandelic acid or cinnamic acid; or with heteroaromaticcarboxylic acids, such as nicotinic acid; or with aliphatic or aromaticsulfonic acids, such as methanesulfonic acid or toluenesulfonic acid.Dermatologically tolerated salts, in particular salts with acetic acidand/or lactic acid, are preferred.

The compounds of general formula (I) also comprise the possible isomericforms as well as mixtures thereof, e.g. racemic mixtures and mixtures ofrotamers.

The amino acids mentioned in formula (I) can have an L or Dconfiguration, or represent a mixture of both configurations.

In particular, Thr may also represent the isomeric forms allo-Thr, D-Thror D-allo-Thr or a mixture of Thr with D-Thr or allo-Thr with D-allo-Thrat the respective position in the sequence.

Among the compounds of formula (I), the following denotations or thefollowing groups of compounds, respectively, are preferred, wherein

R¹ represents hydrogen, —C(O)—R⁷, —SO₂—R⁷, —C(O)—OR⁷ or —C(O)—N(R⁷)₂,preferably wherein R¹ represents hydrogen, —C(O)—R⁷ or —SO₂—R⁷,preferably hydrogen or —C(O)—R⁷

R² represents, independent of one another, hydrogen or methyl

R³, R⁶ represent, independent of one another, —(CH₂)_(q)—N(R¹)R⁸,preferably, independent of one another, —(CH₂)_(q)—NH₂.

R⁴, R⁵ represent, independent of one another, —CH₂—OR⁸, —CH(CH₃)OR⁸ or—CH₂—CH₂—OR⁸, preferably, independent of one another, —CH₂—OH,—CH(CH₃)OH or —CH₂—CH₂—OH,

R⁷ preferably represents hydrogen, methyl, ethyl, propyl, n-butyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-undecanyl, n-dodecanyl,n-tridecanyl, n-tetradecanyl, n-pentadecanyl, n-hexadecanyl,n-heptadecanyl, n-octadecanyl or n-nonadecanyl, isopropyl, tert.butyl,isobutyl, sec.butyl, isoamyl, phenyl, t-butylphenyl, tolyl, 1- or2-naphthyl, perfluorobutyl, pentadecafluoroheptyl, (+)- or(−)-bornan-2-onyl, 8(Z)-heptadecenyl, 8(Z),11(Z)-heptadecadienyl,4(Z),7(Z),10(Z),13(Z)-nonadecatetraenyl or 8(Z)-11-hydroxyoctadecenyl,1,2-dithiolanyl or C₁-C₁₉-alkyl substituted with amino in ω position, orphenyl optionally substituted with methyl, amino or halogen in ortho-and/or para position, preferably R⁷ as a part of XR⁷ representshydrogen, methyl, ethyl and/or R⁷ as a part of R¹ represents an alkylresidue with 14 to 17 C atoms.R⁸ represents hydrogen, C₁-C₄-alkyl or —C(O)—R⁷, preferably hydrogen ormethyl.X represents oxygen, —NH—, preferably oxygen,XR⁷, with X=O, also represents the esters of α-tocopherol, tocotrienolor retinol or the carboxylic acid (with R⁷=H), preferably the carboxylicacid,m, n, p represent, independent of one another, zero or 1, andq in R³ and R⁶ represents, independent of one another, the integer 1, 2,3 or 4.

R⁷—C(O)— most preferably represents the residue of a saturated orunsaturated fatty acid with 6, 8, 10, 12, 14, 16 or 18 C atoms,preferably the corresponding residue of a saturated fatty acid,preferably the corresponding residue of caprylic acid[CH₃—(CH₂)₆—C(O)—], lauric acid [CH₃—(CH₂)₁₀—C(O)—], myristic acid[CH₃—(CH₂)₁₂—C(O)—], palmitic acid [CH₃—(CH₂)₁₄—C(O)—] and/or stearicacid [CH₃—(CH₂)₁₆—C(O)—].

Representative examples of compounds of formula (I) are listed in Table5 (after Example 7).

The compounds of the present invention can be manufactured according tomethods known per se in peptide chemistry. The preferred procedurecomprises fully assembling the compound of the present invention, e.g. acompound of formula (I), optionally splitting off the remainingprotective group(s) and optionally acylating a free amino group and/orconverting the obtained compound into an acid addition salt and/or anobtained acid addition salt into the corresponding conjugate base orinto another salt.

The compounds of the present invention can be used for the preparationof a dermopharmaceutically and/or cosmetically active composition. Suchcompositions contain an effective quantity of at least one compound ofthe present invention or a salt thereof, in the range from 0.5 ppm to5,000 ppm (w/w), preferably between 1 ppm and 1000 ppm (w/w), calculatedon the weight of the compound of the present invention and of thebulking agent(s). The compounds of the present invention can be used asa solution, a dispersion, an emulsion or encapsulated in carriers, suchas e.g. in macro-, micro- or nanocapsules, in liposomes or chylomicrons,or enclosed in macro-, micro- or nanoparticles or in microfungi oradsorbed on powdered organic polymers, talc, bentonite and furtherinorganic carriers.

The compounds of the present invention can be used in any galenic form,such as emulsions, milks, lotions, ointments, gelatinous and viscous,lifting and emulsifying polymers, pomades, shampoos, soaps, gels,powders, sticks, sprays, body oils, face masks or a plaster fortransdermal application.

The compounds of the present invention can be used with any further,commonly used ingredient, such as extraction lipids and/or syntheticlipids, gelatinous and viscous, lifting and emulsifying polymers, water-or fat-soluble active agents, plant extracts, tissue extracts, marineextracts, sun-protective agents, antioxidants, water-retaining andbarrier substances as well as skin-revitalizing agents.

The compounds of the present invention are used in cosmetic applicationsto enhance wound healing and hydration, and in all skin care products,in particular against the formation and aggravation of wrinkles andagainst all consequences of natural or accelerated (sun rays, pollution)skin aging.

The compounds of the present invention as well as the cosmetic anddermopharmaceutical compositions containing same can be used for thepreparation of a drug to promote wound healing and hydration, and forall skin care products, in particular against the formation andaggravation of wrinkles and against all consequences of natural oraccelerated (sun rays, pollution) skin aging.

The present invention also relates to a method for delaying or treatingskin aging, in particular wrinkle formation, which comprises theapplication on the skin of a compound of the present invention.Analogously, the present invention relates to a method for acceleratingwound healing and/or improving skin hydration, which comprises theapplication on the skin of a compound of the present invention.

The following examples illustrate the invention without limiting itsscope. The following abbreviations are used in the text and in Examples1-7:

-   AcOH: Acetic acid-   Boc: tert.-Butyloxycarbonyl-   Dab: 2,4-Diaminobutyric acid-   Dap: 2,3-Diaminopropionic acid-   DBU: 1,8-Diazabicyclo[5,4,0]undec-7-ene(1,5-5)-   DIPEA: Diisopropylethylamine-   DMEM: Dulbecco's Modified Eagle Medium-   Et: Ethyl-   FCS: Fetal Calf Serum-   HSe: Homoserine-   Hyp: Hydroxyproline-   Gly: Glycine-   Lipoyl: α-D,L-Lipoic acid-   Me: Methyl-   MEM: Minimal Essential Medium-   N-Me-Ser: N-Methyl-Serine-   N-Me-Thr: N-Methyl-Threonine-   NMM: N-Methylmorpholine-   Orn: Ornithine-   Palm: Palmitoyl-   PBS: Phosphate buffered saline-   Pr: Propyl-   RT: Room temperature-   TBTU:    O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-tetrafluoroborate-   tBu: t-Butyl-   TFA: Trifluoroacetic acid-   TGF-β(beta)1: Transforming Growth Factor-beta(β)

Example 1 Determination of the Stimulation of Collagen Synthesis TypeI+III in Fibroblast Cell Cultures (ATCC CCL110) by Treatment with theOligopeptide Derivatives of the Present Invention

Method:

The collagen I and III content in fibroblasts is measured with theSirius Red microassay. The quantitative determination of collagen iscarried out after 24 hours of incubation with the correspondingoligopeptide derivatives. The extracellularly accumulated collagen isdetermined. Vitamin C and TGF-beta-1 are used as positive controls.

Method:

ATCC CCL110 fibroblasts are incubated in culture medium at a density ofapprox. 125,000 cells/well in 6-well cell culture plates for 2 days (37°C./5% CO₂). Afterwards, the medium is discarded and washed twice withPBS, whereupon 1 ml of test medium with corresponding test substances isadded. After incubation with test substance under culture conditions(37° C./5% CO₂) for further 24 hours, the extracellular collagen ismeasured with the Sirius Red assay.

Sirius Red Assay Extracellular:

After the incubation period, the total volume of test medium (1 ml) istransferred into a flat-bottom 24-well plate and dried overnight at 55°C. The samples are fixed with 1 ml/well of Bouin's fluid for 1 hour atroom temperature (RT). The fixing solution is discarded and the platesare washed 2-3 times with water. After drying of the plates, 1 ml ofSirius Red dye reagent is added.

The samples are shaken for 1 hour at RT on a microplate agitator atmoderate intensity. Afterwards, the Sirius Red dye reagent is discardedand the plates are washed with 0.01N HCl. The tinted material isdissolved in 0.3 ml of 0.1N NaOH solution and shaken on a microplateagitator for 1 hour at RT.

200 μl of solution is transferred into a flat-bottom 96-well plate andthe optical density is measured at 540 nm against 0.1N NaOH solution asthe blank.

Material:

Culture medium: MEM+10% FCS+100 IU/ml penicillin+0.1 mg/mlstreptomycin+1 mM non essential amino acids+1 mM Na-pyruvate+2 mML-glutamine.

Test medium: DMEM without phenol red (AMIMED)+100 IU/ml penicillin+0.1mg/ml streptomycin+80 μg/ml beta(β)-aminopropionitrile.

Dilution of the Substances:

Vitamin C 100 mM (SIGMA A4034) is diluted to 50 μg/ml in the testmedium.

Transforming growth factor TGFβ1 1 μg/ml (SIGMA T1654) is diluted to 0.1ng/ml in the test medium.

The oligopeptide derivatives of the present invention are prepared in aregular concentration of 10 mM and diluted to 50 μM in the test medium.

Reagents for the Sirius Red Microassay:

Sirius Red F3BA dye reagent as the stock solution 1 mg/ml in saturatedaqueous picric acid solution. Bouin's fluid: 15 ml of saturated aqueouspicric acid solution+5 ml of formaldehyde+1 ml of acetic acid. Theresults are shown in Table 1.

TABLE 1 Results, quantitative determination of collagen type I + IIIafter 24 hours: Test substance at a concentration of 50 μM except whereotherwise stated Extracellular Control without active 100% TGFβ1 0.1ng/ml 115% Vitamin C 128% H-Dab-Ser-OH × 2 TFA 131%H-Lys-allo-Thr-allo-Thr-Lys-Ser-OH × 3 AcOH 139%H-Lys-Thr-allo-Thr-Lys-Ser-OH × 3 AcOH 134% H-Lys-Thr-Thr-Dab-Ser-OH × 3AcOH 137% Lipoyl-Lys (Lipoyl)-Thr-Thr-Lys (Lipoyl)-Ser-OH 162%Palm-Lys-Thr-Ser-Lys-Ser-OH 128%

Example 2 Determination of the Fibronectin Content after Treatment withthe Oligopeptide Derivatives of the Present Invention

Method:

The fibronectin (laminin or collagen VII) content in the fibroblastcells is determined by immuno-dot blotting. Cultivation and incubationof the cells are carried out in the same way as described in Example 1.Afterwards, the cells are lysed and the lysates (0.5 ml) are blotted ona nitrocellulose sheet in a prepared bio-dot device. The sheets are thenincubated with a specific primary antibody to human fibronectinaccording to the western blotting method. A conjugate with alkalinephosphatase is used as the secondary antibody. The intensity of thebands is visually determined semi-quantitatively after dying andstopping of the reaction. The results are shown in Table 2.

TABLE 2 Results, quantitative determination of fibronectin (or laminin Vor collagen type VII) after 24 hours of incubation: Extracellular Testsubstance, at a concentration of 50 μM (relative dyeing except whereotherwise stated intensity) Control without active ++ TGFβ1 0.1 ng/ml ++Vitamin C +++(+) H-Dab-Ser-OH × 2 TFA ++++H-Lys-allo-Thr-allo-Thr-Lys-Ser-OH × 3 AcOH ++++H-Lys-Thr-Thr-Dab-Ser-OH × 3 AcOH ++++ H-Lys-Thr-allo-Thr-Lys-Ser-OH × 3AcOH +++(+) Lipoyl-Lys (Lipoyl)-Thr-Thr-Lys (Lipoyl)-Ser-OH ++++++Palm-Lys-Thr-Ser-Lys-Ser-OH +++(+)

Example 3 Formulation of an Ointment

Method: Ingredients 1-5 (A) are heated to 70° C. Ingredients 6-7 (B) areheated to 75° C. Under stirring B is added to A, cooled to 50° C.,homogenized and cooled to 30° C. Afterwards, ingredients 8-9 (C) andingredient 10 (D) are added one after the other and stirred cold. Theformulations are shown in Table 3.

TABLE 3 Number Ingredient % w/w 1 (A) Tego Care 450 3.00 2Cetearylalcohol 2.25 3 Glycerylstearate 2.25 4 Cetiol 868 10.00 5Squalane 5.00 6 (B) Deionized water 66.995 7 Sodium hyaluronate 5.00 8(C) Glycerin 5.00 9 Phenonip 0.5 10 (D)Octyl-1-SO₂-Lys-Ser-Ser-Dab-Ser-OH 0.005

Example 4 Formulation of a Gel

Method: Ingredients 2-6 (A) are dissolved one after the other indeionized water. After adjusting the pH to 6.0 with ingredient 7 (B),ingredient 8 (C) is added. The formulations are shown in Table 4.

TABLE 4 Number Ingredient % w/w 1 (A) Deionized water 92.095 21,3-Butanediol 5.00 3 Phenonip 0.50 4 Abil B 8843 1.50 5 CarboxymethylCellulose 0.15 6 Carbopol Ultrez 10 0.75 7 (B) NaOH 8 (C)Octyl-SO₂-Lys-Ser-Ser-Lys-Ser-OH 0.005

Examples 5-7

The following embodiments 5-7 describe the synthesis of the compounds offormula (I) of the present invention and of salts of such compounds. Theeluates and products obtained according to the examples are analysedusing proton NMR, HPLC-electrospray MS or elementary analysis. Thecompounds can be manufactured according to known methods describedhereinafter (general instructions from M. Bodanszky “The Practice ofPeptide Synthesis”, Springer, 2^(nd) Edition, 1994). Accordingly, theamino acid, e.g. serine, is bound to a resin at the carboxy terminal endin a solid-phase synthesis, whereby its amino group is protected by aprotective group, e.g. by the Fmoc protective group. The side chain isprotected with, e.g., Boc or t-butyl. If necessary, the protectivegroups are selectively split off in order to link up the further aminoacid derivatives with the reagents commonly used in peptide synthesisuntil the desired chain is completely built up. Afterwards, the peptideor peptide analog, respectively, is split off from the resin at thecarboxy terminal end and this carboxy terminal end is connected withvarying C(O)—R⁷, SO₂—R⁷, C(O)—OR⁷ or C(O)—N(R⁷)₂ residues, whereupon theprotective groups are removed.

Example 5 H-Lys-Thr-Ser-Orn-Ser×3 TFA

Synthesis of H-Lys(Boc)-Thr(tBu)-Ser(tBu)-Orn(Boc)-Ser(tBu)-OH×TFA (5a):

In a typical solid-phase synthesis protocol, the pentapeptide isobtained by repetitive coupling of 18.7 g (14.0 mmol, charge: 0.75mmol/g) of commercial H-Ser(tBu)-chlorotrityl resin with 16.8 mmol ofthe amino acids Fmoc-Orn(Boc)-OH, Fmoc-Thr(tBu)-OH (2×),Fmoc-Lys(Boc)-OH, 18 mmol TBTU, 33.6 mmol collidin and unblocking with1% DBU in DMF (2×5 min), splitting from the resin with 1% TFA indichloromethane and purification over Sephadex LH20®, yield: 9.3 g(64%).

Synthesis of H-Lys-Thr-Ser-Orn-Ser-OH×3TFA (5b):

9.3 g of 5a is stirred for 1 hour at RT in a mixture composed of 59 mlof TFA, 1.25 ml of water and 1.25 ml of triisopropylsilan. Afterreduction to ⅓ of the volume, precipitation with diethylether is carriedout, yield: 6.0 g (76%).

Example 6

Synthesis of CH₃—(CH₂)₇—SO₂-Lys-Thr-Ser-Orn-Ser×2 TFA (6):

2 g (2.2 mmol) of 5a is dissolved in DMF (20 ml) and stirred with 0.48 g(2.3 mmol) of 1-octanesulfochloride and 0.48 g (4.0 mmol) of DIPEA for10 h at RT. After evaporation of the solvent, the crude product isstirred with 59 ml of TFA, 1.25 ml of water and 1.25 ml oftriisopropylsilan for 1 h at RT. After reduction to ⅓ of the volume,precipitation with diethylether and purification over Sephadex LH20® arecarried out, yield: 1.4 g (70%).

The peptide or conjugate can also be protonized with an inorganic acid,e.g. HCl, HBr, H₂SO₄ or H₃PO₄, or with an organic acid, e.g. formicacid, oxalic acid or tartaric acid, leading to corresponding salts of 6.

Example 7 H-Dab-Ser-OH×2 AcOH

According to the described solid-phase protocol from Example 5a, theobtained H-Dab-Ser-OH×2 TFA (3.0 g, 6.90 mmol) is analogouslytransformed in the acetate salt over an ion exchanger, yield 2.0 g(90.0%).

The following compounds can also be synthesized according to the methodsdescribed in Examples 5-7:

TABLE 5 No Sequence ESI-MS  1 H-Dap-Ser-OH × 2TFA 192.1  2Palm-Orn-Ser-OH × TFA 458.5  3 Palm-Orn-N-Me-Ser-OH × TFA  4Palm-Dab-Ser-OH × TFA 444.3  5 Laurinoyl-Lys-Ser-OH × TFA 416.5  6Palm-Lys-Ser-OH × TFA 472.7  7 Oleoyl-Lys-Ser-OH × TFA 498.6  8Palm-Lys-Thr-Thr-Dap-Ser-OH × 2AcOH 760.8  9 Palm-Lys-Thr-Thr-Dab-Ser-OH× 2AcOH 775.0 10 Laurinoyl-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 732.8 11Myristinoyl-Lys-Thr-Thr-Orn-Ser-OH × 760.8 2AcOH 12Stearinoyl-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 817.1 13Palmitoleinoyl-Lys-Thr-Thr-Orn-Ser-OH × 787.1 2AcOH 14Oleoyl-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 815.0 15Eicosaenoyl-Lys-Thr-Thr-Orn-Ser-OH × 843.1 2AcOH 16Palm-Lys-Thr-Thr-Orn-Thr-OH × 2AcOH 803.1 17 Palm-Lys-Ser-Ser-Orn-Ser-OH× 2AcOH 761.0 18 Octadecyl-NH-C(O)-Lys-Ser-Ser-Orn-Ser- 846.1 OH × 2AcOH19 Hexadecyl-NH-C(O)-Lys-Ser-Ser-Orn-Ser- 818.1 OH × 2AcOH 20Tetradecyl-NH-C(O)-Lys-Ser-Ser-Orn-Ser- 790.1 OH × 2AcOH 21Palm-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 788.9 22Palm-Lys-Thr-Thr-Orn-N-Me-Ser-OH × 2AcOH 23Palm-Lys-Thr-Thr-N-Me-Orn-Ser-OH × 2AcOH 24Palm-Lys-Thr-N-Me-Thr-Orn-Ser-OH × 2AcOH 25Palm-Lys-N-Me-Thr-Thr-Orn-Ser-OH × 2AcOH 26Palm-N-Me-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 27Palm-Lys-N-Et-Thr-Thr-Orn-Ser-OH × 2AcOH 28Palm-Lys-N-Pr-Thr-Thr-Orn-Ser-OH × 2AcOH 29C₈H₁₇-SO₂-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 726.8 30C₁₆H₃₃-SO₂-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 839.1 31C₇F₁₅-C(O)-Lys-Thr-Thr-Orn-Ser-OH × 2AcOH 946.8 32H-Lys-Thr-Thr-Orn-Ser-ORetinyl 3 × 2AcOH

1. A composition comprising an acetate salt selected from the groupconsisting of: Octadecyl-NH—C(O)-Lys-Ser-Ser-Orn-Ser-OH×2AcOH,Hexadecyl-NH—C(O)-Lys-Ser-Ser-Orn-Ser-OH×2AcOH,Tetradecyl-NH—C(O)-Lys-Ser-Ser-Orn-Ser-OH×2AcOH, andPalm-Lys-Ser-Ser-Orn-Ser-OH×2AcOH.
 2. A composition comprising anacetate salt selected from the group consisting of:Palm-Lys-Thr-Thr-Dap-Ser-OH×2AcOH and Palm-Lys-Thr-Thr-Dab-Ser-OH×2AcOH.3. A composition comprising an acetate salt selected from the groupconsisting of: Laurinoyl-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Myristinoyl-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Stearinoyl-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Palmitoleinoyl-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Oleoyl-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Eicosaenoyl-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Palm-Lys-Thr-Thr-Orn-Thr-OH×2AcOH, Palm-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Palm-Lys-Thr-Thr-Orn-N-Me-Ser-OH×2AcOH,Palm-Lys-Thr-Thr-N-Me-Orn-Ser-OH×2AcOH,Palm-Lys-Thr-N-Me-Thr-Orn-Ser-OH×2AcOH,Palm-Lys-N-Me-Thr-Thr-Orn-Ser-OH×2AcOH,Palm-N-Me-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,Palm-Lys-N-Et-Thr-Thr-Orn-Ser-OH×2AcOH,Palm-Lys-N-Pr-Thr-Thr-Orn-Ser-OH×2AcOH,C₈H₁₅—SO₂-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,C₁₆H₃₃—SO₂-Lys-Thr-Thr-Orn-Ser-OH×2AcOH,C₈F₁₅—C(O)-Lys-Thr-Thr-Orn-Ser-OH×2AcOH, andH-Lys-Thr-Thr-Orn-Ser-ORetinyl×3AcOH.
 4. A method for increasing theproduction of collagen and fibronectin in human skin by theadministration of a composition according to claim
 1. 5. Adermopharmaceutically and/or cosmetically active composition comprisingat least one acetate salt according to claim 1, present in a quantityranging from 0.5 ppm to 5,000 ppm (w/w), calculated on the weight of thecompound of the present invention and of the bulk material(s).
 6. Acomposition according to claim 5, wherein the composition is in a formselected from the group consisting of a solution, a dispersion, anemulsion and encapsulated in carriers, said carriers selected from thegroup consisting of macrocapsules, microcapsules or nanocapsules,liposomes, chylomicrons, enclosed in macro-, micro- or nanoparticles orin microfungi, and adsorbed on powdered organic and/or inorganicpolymers.
 7. A composition according to claim 5, wherein the compositionis in a form selected from the group consisting of an emulsion, a milk,a lotion, an ointment, a gelatinous and viscous, lifting and emulsifyingpolymer, a pomade, a shampoo, a soap, a gel, a powder, a stick, a spray,a body oil, a face mask and a plaster for transdermal application.
 8. Acomposition according to claim 5, wherein the composition containscommonly used ingredients selected among the group consisting of:extraction lipids and/or synthesis lipids, gelatinous and viscous,lifting and emulsifying polymers, water- or fat-soluble active agents,plant extracts, tissue extracts, marine extracts, sun protection agents,antioxidants, moisturizers and barrier agents and/or skin-revitalizingagents.
 9. A composition according to claim 5, wherein the compositionis a dermopharmaceutically and/or cosmetically active agent thatincreases the production of collagen and fibronectin in human skin. 10.A method for increasing the production of collagen and fibronectin inhuman skin by the administration of a composition according to claim 2.11. A dermopharmaceutically and/or cosmetically active compositioncomprising at least one acetate salt according to claim 2, present in aquantity ranging from 0.5 ppm to 5,000 ppm (w/w), calculated on theweight of the compound of the present invention and of the bulkmaterial(s).
 12. A composition according to claim 11, wherein thecomposition is in a form selected from the group consisting of asolution, a dispersion, an emulsion and encapsulated in carriers, saidcarriers selected from the group consisting of macrocapsules,microcapsules or nanocapsules, liposomes or chylomicrons, enclosed inmacro-, micro- or nanoparticles or in microfungi, and adsorbed onpowdered organic and/or inorganic polymers.
 13. A composition accordingto claim 11, wherein the composition is in a form selected from thegroup consisting of an emulsion, a milk, a lotion, an ointment, agelatinous and viscous, lifting and emulsifying polymer, a pomade, ashampoo, a soap, a gel, a powder, a stick, a spray, a body oil, a facemask and a plaster for transdermal application.
 14. A compositionaccording to claim 11, wherein the composition contains commonly usedingredients selected among the group consisting of: extraction lipidsand/or synthesis lipids, gelatinous and viscous, lifting and emulsifyingpolymers, water- or fat-soluble active agents, plant extracts, tissueextracts, marine extracts, sun protection agents, antioxidants,moisturizers and barrier agents and/or skin-revitalizing agents.
 15. Acomposition according to claim 11, wherein the composition is adermopharmaceutically and/or cosmetically active agent that increasesthe production of collagen and fibronectin in human skin.
 16. A methodfor increasing the production of collagen and fibronectin in human skinby the administration of a composition according to claim
 3. 17. Adermopharmaceutically and/or cosmetically active composition comprisingat least one acetate salt according to claim 3, present in a quantityranging from 0.5 ppm to 5,000 ppm (w/w), calculated on the weight of thecompound of the present invention and of the bulk material(s).
 18. Acomposition according to claim 17, wherein the composition is in a formselected from the group consisting of a solution, a dispersion, anemulsion or encapsulated in carriers said carriers selected from thegroup consisting of macrocapsules, microcapsules or nanocapsules,liposomes or chylomicrons, enclosed in macro-, micro- or nanoparticlesor in microfungi, and adsorbed on powdered organic and/or inorganicpolymers, talc or bentonite.
 19. A composition according to claim 17,wherein the composition is in the form of an emulsion, a milk, a lotion,an ointment, a gelatinous and viscous, lifting and emulsifying polymer,a pomade, a shampoo, a soap, a gel, a powder, a stick, a spray, a bodyoil, a face mask or a plaster for transdermal application.
 20. Acomposition according to claim 17, wherein the composition containscommonly used ingredients selected among the group consisting of:extraction lipids and/or synthesis lipids, gelatinous and viscous,lifting and emulsifying polymers, water- or fat-soluble active agents,plant extracts, tissue extracts, marine extracts, sun protection agents,antioxidants, moisturizers and barrier agents and/or skin-revitalizingagents.
 21. A composition according to claim 17, wherein the compositionis a dermopharmaceutically and/or cosmetically active agent thatincreases the production of collagen and fibronectin in human skin.